In molecular biology, the term restriction fragment length polymorphism (or RFLP, often pronounced "rif-lip") is used in two related contexts: as a characteristic of DNA molecules (arising from their differing nucleotide sequences) by which they may be distinguished and as the laboratory technique which uses this characteristic to compare DNA molecules. The technique is utilized in genetic fingerprinting and paternity testing.

Usually, DNA from an individual specimen is first extracted and purified. Purified DNA may be amplified by polymerase chain reaction (PCR). The DNA is then cut into restriction fragments using suitable endonucleases, which only cut the DNA molecule where there are specific DNA sequences, termed recognition sequence or restriction sites, that are recognized by the enzymes. These sequences are specific to each enzyme, and may be either four, six, eight, ten or twelve base pairs in length. The more base pairs there are in the restriction site, the more specific it is and the lower the probability that it will find a place to be cut. It could be a sticky end cut, where one strand of the double-stranded DNA is left longer than the other, or a blunt end cut, where both strands are cut to the same length. The restriction fragments are then separated according to length by agarose gel electrophoresis. Electrophoresis separates the DNA molecules based on their molecular weight. The resulting gel may be enhanced by Southern blotting. Alternatively, fragments may be visualised by pre-treatment or post-treatment of the agarose gel, using methods such as ethidium bromide staining or silver staining respectively.

The distance between the locations cut by restriction enzymes (the restriction sites) varies between individuals, due to insertions, deletions or transversions. This causes the length of the fragments to vary, and the position of certain amplicons differs between individuals (thus polymorphism). This can be used to genetically tell individuals apart. It can also show the genetic relationship between individuals, because children inherit genetic elements from their parents. Mitochondrial DNA RFLP analyses can lead to the determination of maternal relationships. Fragments may also be used to determine relationships among and between species by comparison of the resulting haplotypes.
RFLP is a technique used in marker assisted selection . Terminal Restriction Fragment Length Polymorphism (TRFLP or sometimes T-RFLP) is a molecular biology technique initially developed for characterizing bacterial communities in mixed-species samples. The technique has also been applied to other groups including soil fungi.

The technique works by PCR amplification of DNA using primer pairs that have been labeled with fluorescent tags. The PCR products are then digested using RFLP enzymes and the resulting patterns visualized using a DNA sequencer. The results are analyzed either by simply counting and comparing bands or peaks in the TRFLP profile, or by matching bands from one or more TRFLP runs to a database of known species.